RU2740013C2 - Method for infertility prediction in females with hypothalamic syndrome - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a method for prediction of infertility in females with hypothalamic syndrome, consisting in that in peripheral blood of women of reproductive age with hypothalamic syndrome, the levels of pro-inflammatory cytokines are simultaneously determined: interleukin-1 beta—IL-1β, interleukin-6—IL-6, interleukin-8—IL-8, tumor necrosis factor—alpha—TNF-α, interferon—gamma—IFN-g and levels of anti-inflammatory cytokines: interleukin-4—IL-4, interleukin-10—IL-10 depending on the menstrual cycle, namely on 5th and 21st days and depending on the body weight index—BWI is more than or equal to 30, and at cytokine levels on 5th day of the menstrual cycle: IL-1β from 2.7 to 3.9 pg/ml, IL-6 from 5.5 to 6.8 pg/ml, IL-8 from 2.9 to 3.7 pg/ml, TNF-α from 3.9 to 4.8 pg/ml, IFN-g from 3.1 to 4.5 pg/ml, IL-4 from 2.8 to 3.3 pg/ml, IL-10 from 5.1 to 6.9 pg/ml, and on 21st day of the menstrual cycle at the levels IL-1β from 2.9 to 4.3 pg/ml, IL-6 from 6.2 to 7.6 pg/ml, IL-8 from 3.2 to 4.1 pg/ml, TNF-α from 4.9 to 5.7 pg/ml, IFN-g from 3.3 to 4.9 pg/ml, IL-4 from 2.9 to 3.6 pg/ml, IL-10 from 5.3 from 7.4 pg/ml, infertility in females with hypothalamic syndrome is predicted.

EFFECT: technical result consists in prediction of infertility in females of reproductive age with hypothalamic syndrome before pregnancy planning.

1 cl, 2 tbl, 2 ex, 3 dwg

Description

The invention relates to clinical medicine, in particular to the specialty of obstetrics and gynecology, and concerns the search for new possibilities for predicting infertility in women of reproductive age with hypothalamic syndrome.

Currently, one of the main factors leading to a decrease in fertility is the deterioration of the reproductive health of women. Infertility is the most important medical and social problem not only in Russia, but also in the countries of Europe and America. In recent years, there has been a steady increase in neuroendocrine pathology. Up to 18-25% of women of reproductive age suffer from hypothalamic syndrome. In most cases, without treatment, the hypothalamic syndrome transforms into a neuroendocrine syndrome, against the background of which 25-72% of women develop infertility. In this regard, it is necessary to search for new methods for predicting reproductive disorders in hypothalamic syndrome in women (The role of immune factors in the functioning of the reproductive system of women / N.O. Motovilova, I.Yu. Kogan, Areg A. Totolyan // Ural Medical Journal. - 2010. - No. 4. - S. 117-121).

As a result of the conducted patent information search, an analogue was found "Method for predicting the development of reproductive disorders" (RF Patent No. 2495431, publ. 10.10.2013), characterized in that cervical discharge in women is examined on the tenth day of the menstrual cycle, parameters are determined levels of interleukin 1 beta and interferon gamma simultaneously. When the content of interleukin 1 beta is more than 38.8 ± 3.8 pg / ml and the simultaneous content of interferon gamma is more than 8.1 ± 0.5 pg / ml, the formation of reproductive disorders is predicted.

The disadvantage of this method is that the study is carried out in women with sexually transmitted infections, which already implies the presence of an inflammatory process at the level of the cervical canal, and interleukin 1 beta and interferon gamma are used to characterize local immunity in the inflammatory process, emphasizing the infectious nature of reproductive disorders functions.

A known method for predicting reproductive function in girls with hypothalamic syndrome of puberty (RF Patent No. 2438136, published on December 27, 2011). The essence of the method lies in the fact that before and after treatment, the level of glycodelin is determined in the menstrual blood. With a glycodelin value of 11.12 ng / ml or more after treatment, pathogenetic therapy is assessed as effective.

The disadvantage of this method is that the marker used for prediction is a marker of the protein-forming function of the endometrium and does not reflect the state of pro- and anti-inflammatory reactions in the endometrium, the study is carried out in the pubertal period in menstrual blood.

A known method for predicting reproductive disorders in women with overweight and obesity (RF Patent No. 2617491, publ. 25.04.2017 g). The essence of the proposed method lies in the fact that early prediction is achieved by determining the activity of NF-kB in the aspirate from the uterine cavity, with an unfavorable prognosis for pregnancy - NF-kB> 6.86 pg / ml.

The disadvantage of this method is that the modulating processes of apoptosis, angiogenesis, differentiation and proliferation are determined at the local level, at the endometrial level, but does not reflect the state of immunity, in particular pro- and anti-inflammatory reactions at the systemic level.

Known methods for determining the various mechanisms of development of primary and secondary infertility in women: Kulakov V.I., Prilepskaya V.N., Radzinsky V.E. // Guidelines for outpatient care in obstetrics and gynecology. M .: GEOTAR-Media, - 2007. - S. 849-855 / Krutova V.A., B.G. Ermoshenko A.M. Chulkova, Possibilities for predicting the development of female infertility // Kuban Scientific Medical Bulletin. - 2009. - No. 9 (114). - S. 73-80 /. Zhukovets I.V., Leshchenko O.Ya., Atalyan A.V. The main mechanisms of primary infertility in women of reproductive age with hypothalamic dysfunction during puberty Acta Biomedica scietifica - 2017, - Volume 2, - No. 5, Part 1. - P. 15-19 /.

The disadvantage of these studies is that they do not reflect the degree of pro- and anti-inflammatory reactions at the systemic level in women with infertility.

A known method for predicting reproductive disorders in women of reproductive age with hypothalamic dysfunction / RF Patent No. 2617247, published 04.24.2017 /. The essence of the method lies in the fact that early prediction is achieved through the simultaneous assessment of pro- and anti-inflammatory cytokines (TNF-α and IL-10) in aspirate from the uterine cavity. With an unfavorable prognosis for pregnancy, TNF-α> 20.2 pg / ml, IL-10 <4.78 pg / ml.

The disadvantage of this method is the determination of one pro and one anti-inflammatory interleukins at the local level, on the 21st day of the menstrual cycle, which does not allow a comprehensive assessment of all links of immunity and timely (with the onset of the menstrual cycle) to carry out preventive and therapeutic measures before the pregnancy planning stage.

It is known that the normal development and functioning of the reproductive system is carried out with close interaction of the immune and endocrine systems. Macrophages play an important role as mediators in the interaction of the immune and endocrine systems. When stimulated, they secrete interleukin - 1, interleukin - 6, tumor necrosis factor, Interleukin - 1 acts on the hypothalamic region. Interleukin - 6 stimulates the hypothalamic-pituitary-adrenal system. Interleukin - 8 is involved in the processes of ovulation, menstruation, fertilization and embryo implantation. Interferon - gamma blocks the development of type 2 T-helpers, inhibiting the synthesis of interleukin - 4, and together with estradiol increases the number of intraepithelial lymphocytes and polymorphonuclear lymphocytes in the endometrium. Interleukin - 10 has a powerful anti-inflammatory and immunomodulatory effect. It suppresses excess production of pro-inflammatory mediators activated by macrophages and type 1 T-helper cells. The level and quantitative ratio of cytokines changes depending on the phase of the menstrual cycle (Gadieva FG The relationship of the immune and endocrine systems in women of reproductive age // Obstetrics and gynecology. - 2001. - No. 1. - S. 11-13). According to modern concepts, a large number of signaling substances are synthesized in white adipose tissue, including tumor necrosis factor (TNF-α), interleukin - 6 (IL-6), interleukin - 8 (IL-8) (Petunina N.A., Makarov I.O., Trukhina L.V., Pchelintseva N.A., Kuzina I.A.Hormones of adipose tissue and their role in reproductive dysfunction in obese women // Obesity and metabolism. - 2011. - No. 4 - С . 9-14). In addition, given that different cells can be the source of synthesis of the same cytokine, when interpreting the cytokine profile data, it is necessary to evaluate not each indicator separately, but groups of indicators.

The proposed invention is aimed at an earlier prediction of infertility in women of reproductive age with hypothalamic syndrome.

The technical result is achieved by the fact that in the peripheral blood of women of reproductive age with hypothalamic syndrome, the levels of pro-inflammatory cytokines are simultaneously determined: interleukin - 1 beta - IL-1β, interleukin - 6 - IL-6, interleukin - 8 - IL-8, tumor necrosis factor - alpha-TNF-α, interferon - gamma - IFN-g and levels of anti-inflammatory cytokines: interleukin - 4 - IL-4, interleukin - 10 - IL-10, depending on the day of the menstrual cycle, namely on days 5 and 21 and depending on from body mass index - BMI is greater than or equal to 30, and with cytokine levels on the 5th day of the menstrual cycle: IL-1β from 2.7 to 3.9 pg / ml, IL-6 from 5.5 to 6.8 pg / ml, IL-8 from 2.9 to 3.7 pg / ml, TNF-α from 3.9 to 4.8 pg / ml, IFN-g from 3.1 to 4.5 pg / ml, IL- 4 from 2.8 to 3.3 pg / ml, IL-10 from 5.1 to 6.9 pg / ml, and on day 21 of the menstrual cycle at IL-1β levels from 2.9 to 4.3 pg / ml , IL-6 from 6.2 to 7.6 pg / ml, IL-8 from 3.2 to 4.1 pg / ml, TNF-α from 4.9 to 5.7 pg / ml, IFN-g from 3.3 to 4.9 pg / ml, IL-4 2.9 to 3.6 pg / ml, IL-10 from 5.3 to 7.4 pg / ml predict infertility in women with hypothalamic syndrome.

The proposed method is carried out as follows.

Blood is taken from a vein in women with a body mass index - BMI greater than or equal to 30 using a disposable syringe on the 5th and 21st days of the menstrual cycle. To obtain serum, a test tube with blood is left in an inclined (at an angle of 10 ° - 20 °) at room temperature for 30 minutes to form a clot, after which the test tube with blood is shaken (to separate the clot from the tube wall), closed with a stopper and set overnight in the refrigerator at a temperature of + 4 ° C. After separating the serum from the clot, it was centrifuged at 3000 g for 7-10 min. Then the serum was aspirated with a pipette into sterile plastic epindorfs and stored at -20 ° C until ELISA.

The levels of pro-inflammatory cytokines interleukin - 1 beta IL-1β, interleukin - 6 IL-6, interleukin - 8 IL-8, tumor necrosis factor - alpha TNF-α, interferon - gamma IFN-g and anti-inflammatory interleukin - 4 IL-4, interleukin - 10 IL-10 was determined using the kits "Interleukin 1 - IFA-BEST", "alpha-TNF - IFA-BEST", "Interleukin 6-IFA-BEST", "Interleukin 8 - IFA-BEST", "Interleukin 4 - IFA-BEST "," Interleukin 10 - IFA-BEST "," gamma-Interferon - IFA-BEST "(JSC" Vek-tor-Best ", Novosibirsk), according to the protocols for setting ELISA from the manufacturer.

New features of the claimed method are:

- prediction of infertility before planning pregnancy;

- assessment of groups of pro- and anti-inflammatory cytokines, as well as interferon in the peripheral blood;

- assessment of groups of pro- and anti-inflammatory cytokines, as well as interferon in the peripheral blood in women with hypothalamic syndrome and body mass index equal to 30 or more;

- simultaneous determination of groups of pro-inflammatory (IL-1β, IL-6, IL-8, TNF-α), interferon-gamma (IFN-g) and anti-inflammatory (IL-4, IL-10) cytokines;

- venous blood is used as a biological material (systemic level of immunity regulation);

- predicting infertility of pregnancy in patients with hypothalamic syndrome, when determining the level of cytokines in the venous blood on the 5th day and 21st day of the menstrual cycle, which makes it possible to timely carry out therapeutic and prophylactic measures.

The proposed method is based on a new direction, obtained through laboratory research: changes in the content of indicators of innate and humoral immunity links, namely pro-inflammatory cytokines IL-1β, IL-6, IL-8, TNF-α and IFN-g, as well as anti-inflammatory cytokines IL-4, IL-10 in women of reproductive age with hypothalamic syndrome compared with women of reproductive age who had no disorders in the hypothalamic-pituitary system (conditionally healthy).

For the first time, the author discovered that in women of reproductive age with hypothalamic syndrome in the peripheral blood, on the 5th day of the menstrual cycle, there is an increase in the proinflammatory cytokines IL-1β, IL-6, IL-8, TNF-α, IFN-g, and an increase in anti-inflammatory cytokines IL-4, IL-10. On day 21 of the cycle, an increase in the concentration of pro-inflammatory cytokines IL-1β, TNF-α, IFN-g, a significant increase in anti-inflammatory cytokines IL-4, IL-10 and pro-inflammatory cytokines IL-6, IL-8.

The study was conducted in 68 women of reproductive age with hypothalamic syndrome and 30 conditionally healthy women from 2010 to 2018 on the basis of antenatal clinics and antenatal clinics No. 1 of the City Polyclinic No. 8 named after N.I. Pirogov ", Astrakhan.

When assessing the levels of pro-inflammatory cytokines on the 5th day of the menstrual cycle, there is a significant increase in IL-1β in relation to the control (p <0.05), also IL-6 (p <0.001), TNF-α (p <0.05) , IFN-γ (p <0.05), IL-8 (p <0.05). The concentration of anti-inflammatory cytokines IL-4 (p <0.05) IL-10 (p <0.001) in the peripheral blood also tended to increase. The analysis of the levels of all cytokines is studied, and showed their significant increase from the 5th to the 21st day of the menstrual cycle. The described trend is reflected in Table 1, 2 and Fig.

where: fig. 1 - dynamics of concentrations of TNF-α and IFN-g in the blood serum of the examined;

fig. 2 - dynamics of levels of IL-1β, IL-6, IL-8 in the blood serum of the examined;

fig. 3 - dynamics of serum concentrations of the examined IL-4, IL-10.

Below are the results of testing.

Example 1.

Patient G., 22 years old, complaints at the time of examination: irregular menstruation, lack of pregnancy for 2 years with regular sex without contraception.

History: menstruation from 11 years, for 4-6 days, after 30-35 days, from 14 years - after 40-60 days, irregular. In the reproductive age, she did not go to a doctor-gynecologist. In the puberty period, she turned to the children's gynecologist at the Children's City Polyclinic No. 1 JSC. After the examination, the diagnosis was made: Dysfunction of the hypothalamus of puberty. Secondary oligomenorrhea.

Sexual life since 20 years of marriage, without contraception. There was no pregnancy. Objectively: BMI 34.5 kg / m ^2, striae pearlescent colors in the hips and buttocks, the mammary glands. Clinical diagnosis: Secondary oligomenorrhea. Obesity 1 tbsp. Complicated gynecological anamnesis (hypothalamic dysfunction during puberty). The examination was carried out: blood was taken from a vein and indicators of pro-inflammatory and anti-inflammatory cytokines were determined on the 5th day of the menstrual cycle: IL-1β - 2.7 pg / ml, IL-6 - 5.7 pg / ml, IL-8 - 3.0 pg / ml, TNF-α - 4.3 pg / ml, IFN-g - 3.2 pg / ml IL-4 - 2.8 pg / ml, IL-10 - 5.2 pg / ml, on day 21 of the menstrual cycle IL-1β - 4.2 pg / ml, IL-6 - 6.9 pg / ml, IL-8 - 3.8 pg / ml, TNF-α - 5.2 pg / ml, IFN -g - 4.6 pg / ml IL-4 - 3.6 pg / ml, IL-10 - 7.2 pg / ml.

Based on the data obtained, infertility is predicted.

After the examination, the patient underwent pathogenetic therapy for 7 months with recommendations on the use of barrier methods of contraception. Pregnancy occurred 6 months after the treatment. It ended with the birth of a full-term baby.

Example 2.

Patient L., 25 years old.

Complaints at the time of examination: does not show.

History: menstruation from 12 years, for 5-7 days, after 45-90 days, irregular. She has been taking combined oral contraceptives for 2 years. Sexual life since 21 years. Before taking combined oral contraceptives, she was not protected from pregnancy. Pregnancy did not occur. In the puberty period, she turned to the children's gynecologist at the Children's City Polyclinic No. 1 JSC. After the examination, the diagnosis was made: Dysfunction of the hypothalamus of puberty. Primary oligomenorrhea. At the age of 23, she turned to the antenatal clinic at the place of residence, where combined oral contraceptives were prescribed in order to regulate the menstrual cycle. There was no pregnancy.

Objectively: BMI - 33.9 kg / m ² , pearlescent striae in the thighs and buttocks, mammary glands.

Clinical diagnosis: Primary oligomenorrhea. Complicated gynecological anamnesis (hypothalamic dysfunction during puberty).

The patient was advised to discontinue combined oral contraceptives. A month later, after the abolition of combined oral contraceptives, a survey was carried out: blood was taken from a vein and the content of pro-inflammatory and anti-inflammatory cytokines on the 5th day of the menstrual cycle was determined: IL-1β - 2.8 pg / ml, IL-6 - 5.6 pg / ml, IL-8 - 3.1 pg / ml, TNF-α - 4.2 pg / ml, IFN-g - 3.2 pg / ml IL-4 - 2.8 pg / ml, IL-10 - 5.2 pg / ml, on day 21 of the menstrual cycle IL-1β - 4.0 pg / ml, IL-6 - 6.6 pg / ml, IL-8 - 4.0 pg / ml, TNF-α - 5.0 pg / ml, IFN-g - 4.7 pg / ml IL-4 - 3.5 pg / ml, IL-10 - 7.1 pg / ml - infertility is predicted.

After the examination, pathogenetic therapy was prescribed in combination with pregravid preparation in accordance with the Order of the Ministry of Health of the Russian Federation of 12.11.12 No. 572n. for 6 months. A month after the treatment, pregnancy began. It ended with the birth of a full-term baby.

The proposed method allows you to predict infertility earlier before planning pregnancy. It also provides the possibility of screening examination of patients with hypothalamic syndrome, timely correction of the identified disorders, which will allow assessing the risk, accelerating the implementation of the generative function and reducing material costs for therapy.

Claims (1)

A method for predicting infertility in women with hypothalamic syndrome, which consists in the fact that in the peripheral blood of women of reproductive age with hypothalamic syndrome, the levels of pro-inflammatory cytokines are simultaneously determined: interleukin - 1 beta - IL-1β, interleukin - 6 - IL-6, interleukin - 8 - IL-8, tumor necrosis factor - alpha - TNF-α, interferon - gamma - IFN-g and levels of anti-inflammatory cytokines: interleukin - 4 - IL-4, interleukin - 10 - IL-10, depending on the day of the menstrual cycle, namely on days 5 and 21 and depending on body mass index - BMI is greater than or equal to 30, and with cytokine levels on the 5th day of the menstrual cycle: IL-1β from 2.7 to 3.9 pg / ml, IL-6 from 5.5 to 6.8 pg / ml, IL-8 from 2.9 to 3.7 pg / ml, TNF-α from 3.9 to 4.8 pg / ml, IFN-g from 3.1 to 4 , 5 pg / ml, IL-4 from 2.8 to 3.3 pg / ml, IL-10 from 5.1 to 6.9 pg / ml, and on day 21 of the menstrual cycle at IL-1β levels from 2, 9 to 4.3 pg / ml, IL-6 from 6.2 to 7.6 pg / ml, IL-8 from 3.2 to 4.1 pg / ml, TNF-α from 4.9 days about 5.7 pg / ml, IFN-g from 3.3 to 4.9 pg / ml, IL-4 from 2.9 to 3.6 pg / ml, IL-10 from 5.3 to 7.4 pg / ml predict infertility in women with hypothalamic syndrome.

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